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Multiple fluorescence immunoassay method for detecting mycoplasma gallisepticum and mycoplasma synoviae

A technology for Mycoplasma gallisepticum and Mycoplasma gallisepticum, which is applied in the directions of biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, to reduce detection costs, avoid cross-hybridization, and achieve good repeatability.

Inactive Publication Date: 2016-12-14
GUANGDONG LAB ANIMALS MONITORING INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the detection of Mycoplasma gallisepticum and Mycoplasma gallisepticum by using multiple immunofluorescence analysis method

Method used

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  • Multiple fluorescence immunoassay method for detecting mycoplasma gallisepticum and mycoplasma synoviae
  • Multiple fluorescence immunoassay method for detecting mycoplasma gallisepticum and mycoplasma synoviae
  • Multiple fluorescence immunoassay method for detecting mycoplasma gallisepticum and mycoplasma synoviae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Primer Design

[0053] Design a group of multiple fluorescence immunoassay primers for detecting Mycoplasma gallisepticum and Mycoplasma gallisepticum wherein, the primer pair for detecting Mycoplasma gallisepticum is designed according to the conserved sequence of the PVPA gene of Mycoplasma gallisepticum; The primer pair for detecting Mycoplasma gallinarum is designed according to the conserved sequence of the vlhA gene of Mycoplasma gallinarum. After repeated trials and screening, the following primer sequences were obtained:

[0054] The nucleotide sequence of the primer pair that is used to detect Mycoplasma gallisepticum is as follows:

[0055] MG primer G1: 5'-CTTACAGATTACAGGAGCAG-3' (SEQ ID NO.1),

[0056] MG primer G2: 5'-AAGCATTTCATTTGTTTTAC-3' (SEQ ID NO.2);

[0057] The nucleotide sequence of the primer pair that is used to detect Mycoplasma gallinarum is as follows:

[0058] MS Primer S1: 5'-ATTAGCAGCTAGTGCAGTGG-3' (SEQ ID NO.3),

[0059] MS ...

Embodiment 2

[0063] Example 2: Establishment of multiple fluorescent immunoassay kits for detecting MS and MG

[0064] The kit includes the following components:

[0065] (1) The 2-fold fluorescent immunoassay primer set designed in Example 1;

[0066] (2) Two kinds of fluorescently encoded microspheres containing anti-tag sequences encoding different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences on the upstream primers of the multiple fluorescent immunoassay primer set; the two kinds of microspheres The balls were all purchased from luminex company, and the numbers of fluorescently coded microspheres corresponding to MS and MG were MTAG-A025 and MTAG-042, respectively.

[0067] (3) streptavidin-phycoerythrin complex;

[0068] (4) PCR amplification reaction system.

Embodiment 3

[0069] Example 3 Establishment of Multiple Fluorescence Immunoassay Detection Method for MS and MG

[0070] 1. Plasmid construction

[0071] Use Tiangen’s automatic nucleic acid extractor to extract the DNA of MG and MS pathogens, respectively, and perform PCR amplification with the corresponding primers designed in Example 1. The amplified products are detected by agarose gel electrophoresis and purified by gel cutting. The purified cDNA was connected to the pMD-19T vector with a kit from TaKaRa Company, the connected product was transformed into DH5a competent cells, single clones were selected, colony PCR identification was carried out, and the colonies identified as positive bacteria were subjected to plasmid extraction. Send for sequencing.

[0072] 2. Plasmid PCR amplification

[0073] Use specific amplification MS, MG primers for single and double PCR amplification.

[0074] Preparation of upstream primer mixture: mix G1 and S1 at a ratio of 1:1; preparation of downs...

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Abstract

The invention discloses a multiple fluorescence immunoassay method for detecting mycoplasma gallisepticum and mycoplasma synoviae. The multiple fluorescence immunoassay method has the advantages that the operation is simple; a target amplified fragment is obtained through PCR (polymerase chain reaction), then the amplified fragment, fluorescent encoding microspheres, and streptavidin-phycoerythrin are hybridized, and an MFI (mean fluorescence intensity) value is read through a detection instrument, so as to distinguish different types of pathogens; the mycoplasma gallisepticum and mycoplasma synoviae can be simultaneously detected and identified; the specificity is strong, the sensitivity is high, the repeatability is good, and the like; multiple types of molecules with different purposes in the same sample can be simultaneously detected; the flexibility is good, and the type of to-be-detected pathogens can be increased or decreased on the basis according to requirements.

Description

technical field [0001] The invention belongs to the field of pathogen detection in breeding industry, and in particular relates to a multiple fluorescence immunoassay method for detecting mycoplasma gallisepticum and mycoplasma gallisepticum. Background technique [0002] Chicken mycoplasma disease is an infectious disease caused by mycoplasma, which is mainly transmitted through direct contact with respiratory tract infection and egg infection. The most common ones are Mycoplasma gallisepticum (MG) and Mycoplasma synovialum (MS). Mycoplasma gallisepticum mainly causes chronic respiratory symptoms, air sacs, and decreased egg production in chickens, and is the most serious economic loss to the poultry industry among mycoplasmas; Mycoplasma gallisepticum mainly causes joint lesions and subclinical respiratory symptoms in chickens. [0003] Tract symptoms, air sacculitis and joint lesions lead to a significant increase in the waste rate of ketone bodies in broilers. Clinicall...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6804C12Q1/689C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 郭鹏举朱余军丛峰黄韧陈梅丽
Owner GUANGDONG LAB ANIMALS MONITORING INST
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